TechVault
  • When do researchers use random (degenerate, wobble, mixed) base oligos?

    Mixed bases are used in primers to bind to templates that contain variability or a mixture of sequences at the primer binding sites. Mixed bases can also be used to create diversity in clone libraries and in site directed mutagenesis.

    IDT offers random base oligos. Order them by using an upper case letter N or other IUPAC-IUB symbol (see table below). IDT offers two types of randomization, machine mix and hand mix. Machine mix bases are charged at the standard base price for the scale ordered. Hand mixing is done to provide custom ratios of the bases, and incurs an additional charge. When entering your sequence, the “Mixed Base” tab at the bottom of the page lists the IUB symbols and is where custom mix ratios need to be entered.

    Now you can get gBlocks® Gene Fragments that contain up to 18 consecutive variable bases (N or K) for recombinant antibody generation or protein engineering.See www.idtdna.com/gblocks for more information.

  • What quality checks do gBlocks Gene Fragments with variable bases receive?
    The constant regions are gBlocks® Gene Fragments and undergo size verification by capillary electrophoresis and sequence verification by mass spectrometry. The variable regions cannot currently receive complete analysis (using NGS, which would be needed for this analysis, would be cost prohibitive). We rely on a validated process that we have experimentally confirmed by NGS that ensures >80% of DNA species are present in the final material shipped.
  • Where is the "notes" field for oligo ordering?

    The Notes field is still available for use but is no longer visible by default. You can enable item notes using your account preferences:

    Click on My Account

    Under My Account, click Edit User Preferences

    Select True for Enable Item Notes

    Click Save Options

    The Notes field should now appear in the oligo ordering interface:

  • Should I order my gBlocks Gene Fragment with the 5′ phosphate modification?
    Adding the 5′ phosphate modification to gBlocks Gene Fragments is unnecessary for most applications. The 5′ phosphate is required for traditional blunt-end cloning. Do not add 5′ phosphate for Zero Blunt TOPO® cloning (check manufacturer information).
  • How do I design my gBlocks Gene Fragment for restriction cloning?
    Cloning of gBlocks Gene Fragments is similar to cloning a very pure PCR product. For restriction cloning it is important to add 6–8 nt at the ends of the fragment, beyond the restriction recognition sequence. Most restriction enzymes will not cut efficiently, or at all, if the fragment terminates precisely at the restriction site.
  • How do I determine the number and cost of target capture probes I will need to target N genes/gene regions on my NGS custom panel?
    The number of probes you will need to order and their total cost will vary with the set of genes being targeted. To obtain an accurate assessment, use the free, online Custom Capture Design Tool on our website. The tool will determine the probes required to obtain optimal coverage depth and uniformity of your gene set, as well as provide a rudimentary design of the probes. The tool can handle up to 1000 genes or genomic regions, or up to 1 Mb of FASTA file sequencing information. If you need to order probes for a larger set of sequences, please contact xgen@idtdna.com.
  • What does IDT’s browser warning mean?

    Why are you no longer supporting older web browsers?

    We consider several factors when deciding to stop support of a browser, such as how many of our web requests come from the browser, how much staff time is required to support the browser, and what is the general level of support for the browser among other internet content providers.

    What does “stop supporting this browser” mean?

    When we stop supporting a given browser, we will no longer test new or updated web pages on that browser, and can no longer guarantee that the features on our web pages will work well or at all in the browser. Additionally, if we become aware of problems viewing our web pages that occur only with that browser, we will no longer fix them.

    What changed on the IDT website?

    Actually, nothing. We didn’t turn anything on or off. As described above, we simply will no longer test our pages or fix problems in older browsers versions including Internet Explorer 7.

    What does this really mean about my use of the IDT website?

    Practically this means that users with older browsersshould still be able to use the IDT website for ordering and accessing resources; however, as we update our pages, we expect that over a period of time (which could be weeks or months), the ability of to render our pages correctly will degrade.

    What should I do?

    The best course of action is to upgrade your browser to a more current version. Examples of the most current versions of browsers that we support are as follows:

  • Do fluorescently modified oligonucleotides need to be protected from light?
    The main cause of oligonucleotide damage from light comes from the UV radiation in natural light. Fluorescently labeled oligonucleotides are fairly stable when exposed to artificial light. During normal laboratory use, it is not necessary to protect fluorescently labeled oligos from artificial light sources by using brown tubes and/or foil covering.
  • Is there any information on the stability of fluorescently labeled oligos and the fluorescent modifications?

    The stability of fluorescently labeled oligos and fluorophore function is similar to the stability of a standard oligo.  For optimal stability, oligos that are to be stored long term should be stored frozen, at -20°C.  If the oligos are going to be resuspended, storage in a TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5–8.0; such as IDTE) is better than using water (non-DEPC treated). The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures (see the Technical Report, Oligonucleotide Stability Study, for the data).

    With fluorophores, it’s important to remember that exposure to natural light or ozone can negatively impact stability.

  • Should RNA be stored differently than DNA?
    The inherent chemical structure of RNA makes it less stable than DNA. RNases that degrade RNA are also more prevalent than DNases.  As RNA is a great deal more sensitive to degradation compared to DNA, for short term storage we recommend using a neutral to slightly acidic buffer containing a chelator.  However, RNA is most stable when stored as an ethanol precipitate at –80°C, especially when storing RNA oligos long term.  The biggest concern for RNA oligonucleotide storage is avoiding RNase.
  • What is the stability of RNA vs. DNA?

    RNA is inherently less stable than DNA due to its chemical structure. Additionally RNases are more prevalent in standard laboratory conditions than DNases. As even the slightest exposure to RNase can impact RNA stability, IDT has not performed rigorous long term stability studies for RNA.

  • Do fluorescently modified oligonucleotides need to be protected from light?

    The main cause of oligonucleotide damage from light comes from the UV radiation in natural light. The effects of artificial light on oligo stability are not clear. If fluorescently modified oligos will be exposed to sustained light, protective cover from the lighting source (e.g., using brown tubes and/or foil covering) should be considered.

  • What is the stability of fluorescently labeled oligos and their fluorescent modifications?

    The stability of fluorescently labeled oligos and fluorophore function is similar to the stability of a standard oligo.  For optimal stability, oligos that are to be stored long term should be stored frozen, at -20°C.  If the oligos are going to be resuspended, storage in a TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5–8.0; such as IDTE) is better than using water (non-DEPC treated). The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures (see the Technical Report, Oligonucleotide Stability Study, for the data). 

    With fluorophores, it’s important to remember that exposure to natural light or ozone can negatively impact stability. If fluorescently modified oligos will be exposed to sustained light, protective cover from the lighting source (e.g., using brown tubes and/or foil covering) should be considered.

  • What is the Design or Catalog ID # for my DsiRNA?

    The DsiRNA Design or Catalog ID # appears on the DsiRNA ordering webpage and on your DsiRNA spec sheet. IDT uses this sequence identifier to track what gene the DsiRNA targets and the actual sequence ordered. Its nomenclature is designed to provide information about the sequence.

    DsiRNAs designed and ordered since February 2016

        Example Design ID # hs.Ri.BRCA1.13.2:

    • hs” indicates the species, Homo sapiens (human). Similarly, “mm” means Mus musculus (mouse), “rn” means Rattus norvegicus (rat), and “CD” indicates custom design.
    • Ri” indicates this is an RNA interference (DsiRNA) product.
    • BRCA1” indicates the NCBI gene symbol, in this example, the breast cancer associated 1 gene.
    • 13” indicates that this sequence comes from the 13th iteration of the DsiRNA designs. Custom designs have a unique identifying number.
    • 2” indicates that this was the second design in the search results.

    DsiRNAs designed and ordered before February 2016

         Example Catalog ID # HSC.RNAI.N000546.12.6_2nm:

    • “HSC” indicates that it is “Homo sapiens common”, meaning that if there is more than one splice form in RefSeq, this sequence is common to all forms. Similarly, “HSS” means “Homo sapiens specific” to a single splice variant.
    • “RNAI” indicates that this is a DsiRNA product.
    • “N000546” indicates the RefSeq gene accession number, which is NM_000546 (gene symbol TP53) in this example.
    • “12” indicates that this sequence comes from the 12th iteration of the DsiRNA designs.
    • “6” denotes that this was the 6th in the list of 10 predesigned DsiRNAs and identifies which DsiRNA in the predesigned library was ordered.
    • “2nm” indicates that a 2 nmol scale was ordered.
  • Is it OK to resuspend and store oligos in DEPC treated water?

    No, we do not recommend resuspending or storing oligos in DEPC treated water.  DEPC treated water can be acidic which may cause depurination of the oligo resulting in degradation. For optimal stability, oligos that are to be stored long term should be stored frozen, at–20°C.  If the oligos are going to be resuspended, storage in a TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA; such as IDTE) is better than using water. The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures (see Figures 1–3 above). It is ideal to store oligos in the dark. Exposure to UV light should be avoided, and ambient lab light should be minimized, particularly for some types of modified oligos.

    Also, avoid unnecessary freeze thaw cycles by preparing a freezer stock solution (100 µM) and several working aliquots.  Use the IDT Resuspension Calculator under the "SciTools®" tab of the IDT website for help determining volumes to use.

    Please see the Technical Report, Oligonucleotide Stability Study, for data on oligonucleotide storage and a more thorough explanation.

  • How should I store and resuspend modified oligos?

    Most modified oligos can be stored and resuspended just as unmodified oligos. For long term storage, temperature is the most important factor to consider. For long term storage, whether oligos are dried down, or resuspended in water (non-DEPC treated) or TE Buffer (10 mM Tris pH 8.0, 0.1 mM EDTA; such as IDTE), it is optimal to store them frozen at -20°C. For room temperature or 4°C storage, oligos that are resuspended in TE buffer are more stable than dry oligos. Oligos that are stored in water are the least stable. However, at 4°C oligos stored under all of these conditions are stable for at least 60 weeks.

    It is ideal to store oligos in the dark. Exposure to UV light should be avoided, and ambient lab light should be minimized, particularly for some types of modified oligos.

    Please see the Technical Report, Oligonucleotide Stability Study, for data on oligonucleotide storage and a more thorough explanation.

    For resuspension, we recommend making a stock solution and then smaller working aliquots to avoid contamination of the entire stock, and to limit repeated freeze/thaw cycles that can lead to degradation of the oligo. 

    Notes for specific modifications: 
    Oligos containing photocleavable or photolabile modifications such as Bromo-dU, PC Spacer, PC Biotin, and PC Linker, should be kept away from light and are therefore shipped in amber tubes. We recommend storing them in dark tubes or wrap them in foil.

    Also, amine-modified oligos that will be used in NHS ester reactions should not be hydrated in buffers containing amines (such as TRIS), as the amines in the buffer will compete with the amine-modified oligo in the reaction. These amine-modified oligos can instead be resuspended in 0.1 mM EDTA or non-DEPC treated, nuclease-free water and stored at -20°C.

  • Do your oligos have an expiration date?

    There are several factors that influence oligo stability, including the oligo composition and storage conditions. Due to this variability, we do not routinely provide an expiration date. Please contact Technical Support (custcare@idtdna.com) if you have questions regarding oligo stability and storage.

    If you are using an oligo for a commercial use or as a molecular diagnostics product, please contact our Clinical and Commercial Accounts (mailto:GMPinfo@idtdna.com) or Third Party Manufacturing (tpmi-solutions@idtdna.com) representative. These groups can provide a letter containing expiration dates. 

    IDT can provide custom labels that include an oligo expiration date specified by the customer. Please contact customquotes@idtdna.com for more information regarding the quotation process for this service.

  • What are optimum storage conditions for a custom gene?

    Store custom genes dry / lyophilized at -20°C for ideal long term stability. When ready for use, the gene tube should be briefly centrifuged to ensure that the dried DNA pellet is at the bottom of the tube. Resuspend the dry DNA pellet in 20 µL TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5–8.0; such as IDTE) or non-DEPC treated, nuclease free water to achieve an approximate stock concentration of 100 ng/μL. Incubate at room temperature for 30 minutes, and then vortex for 20 seconds. Centrifuge the tube(s) at 10,000 x g for 1 minute. Create aliquots if desired and store resuspended DNAs (short term) at -20°C.

  • Are there any special requirements for the handling and storage of my labeled probes?

     

    Labeled probes should, for the most part, be handled as other custom oligonucleotide sequences. We recommend resuspending probes in TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA), such as or IDTE. Non-DEPC treated nuclease‑free water can be used, although it can limit shelf life. Ideally, for each resuspended probe, create a stock and several aliquots and store them all at –20°C. For long term oligo storage, temperature is the most important factor to consider. For long term storage, whether oligos are dried down, or resuspended in water (non-DEPC treated) or TE buffer, it is optimal to store them frozen.

    For room temperature or 4°C storage, oligos that are resuspended in TE buffer are more stable than dry oligos. Oligos that are stored in water are the least stable. At 4°C oligos stored under all of these conditions are stable for at least 60 weeks.

    Please see the Technical Report, Oligonucleotide Stability Study, for data on oligonucleotide storage and a more thorough explanation.

  • How should I store my oligo?

    For optimal stability, oligos that are to be stored long term should be stored frozen, at –20°C.  If the oligos are going to be resuspended, storage in a TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5–8.0; for example, IDTE) is better than using water (non-DEPC treated). The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures. It is ideal to store oligos in the dark. Exposure to UV light should be avoided, and ambient lab light should be minimized, particularly for some types of modified oligos.

    Please see the Technical Report, Oligonucleotide Stability Study, for data on oligonucleotide storage and a more thorough explanation.